1. Field of the Invention
The present invention relates to a luciferase gene, polypeptide, and mutants thereof. More particularly, the present invention relates to a luciferase gene, polypeptide, and mutants thereof from the firefly Luciola italica. 
2. Brief Description of Art
Bioluminescence is the emission of light from an organic molecule, such as luciferin, which has been oxidized by oxygen or one of its metabolites. The reaction is catalyzed by the luciferase protein (Luc), a naturally occurring protein that is found in beetles, fireflies and other living organisms, to form luciferyl-adenylate (LH2-AMP) from substrates luciferin (LH2) and ATP (eq. 1). Through a multi-step oxidative process, LH2-AMP is converted to excited state oxyluciferin, the light-emitting product (eq. 2-3) (H. H. Seliger et al., Arch. Biochem. Biophys. 88 (1960) 136-141; E. H. White et al., Bioorg. Chem. 1 (1971) 92-122; M. DeLuca; Adv. Enzymol. 44 (1976) 37-68; J.-Y. Koo et al., Proc. Natl. Acad. Sci. USA 75 (1978) 30-33; J. W. Hastings; Bioluminescence, in: Sperelakis, N., (Ed.), Cell Physiology Source Book, Academic Press, New York, 1995, pp. 665-681; K. V. Wood; Photochem. Photobiol. 62 (1995) 662-673).
Luc.LH2-AMP+O2→Luc.AMP.Oxyluciferin*+CO2  (2)Luc.AMP.Oxyluciferin*→Luc+Oxyluciferin+AMP+hυ  (3)
The generation of light from LH2 is highly efficient (Seliger et al., supra) affording great sensitivity for the detection of the luciferase protein using available light measuring technology. Thus, the luciferase gene is extremely suitable for reporter gene applications (S. J. Gould et al., Anal. Biochem. 175 (1988) 5-13) and in vivo bioluminescence imaging (C. H. Contag et al., Annu. Rev. Biomed. Eng. 4 (2002) 235-260). Luciferases have been used to study gene delivery (Y. Taniyama et al., Gene Ther. 9 (2002) 372-380), gene silencing (P. J. Paddison et al., Proc. Natl. Acad. Sci. USA 99 (2002) 1443-1448) and real-time imaging of luciferase expression in live animals (L. F. Greer, III, et al., Luminescence 17 (2002) 43-74).
Currently, luciferase genes from various species, including Photinus pyralis (common North American firefly), Pyrophorus plagiophathalamus (Jamaican click beetle), Luciola mingrelica (European beetle), and Lampyris noctiluca (glow worm), are used to generate luminescent reporter moieties.
Typically, these reporter moieties emit light in the blue to yellow-green range (400-560 nm) or the green to orange range (510-590 nm). However, emission of light at other wavelengths is useful in many applications. For example, light emitted at a wavelength closer to the red range (“red shifted”) is known to be transmitted through live tissue more efficiently than other wavelengths of visible light. Similarly, light emitted at a wavelength closer to the blue range (“blue shifted”) provides increased sensitivity to standard photomultiplier tubes and is important for dual color assays where it is important to maximize the wavelength separation of the signals. By shifting the bioluminescence emission toward the red or blue wavelengths, it is possible to enhance the utility of luciferase genes in in vivo monitoring and gene expression assays.
There are many uses of luciferase known in the art. Luciferase, from various sources, has been used in various assays and reporting capacities. For example, U.S. Pat. No. 6,387,675 discloses the use of the luciferase gene of the elaterid beetle, P. plagiophthalamus, in eukaryotic cells for biosensing. U.S. Pat. No. 6,812,012 discloses a method of using luciferase to assay intracellular ATP, while a method of using a luciferase gene as a reporter gene is disclosed in U.S. Pat. No. 6,495,355.
Expression of luciferase genes has also been shown in the art. For example, U.S. Pat. No. 6,132,983 discloses the expression of luciferase genes in cells of various host organisms, while U.S. Pat. No. 4,968,613 discloses production of luciferase by incorporating a luciferase gene into a vector inserted into E. coli. U.S. Pat. No. 5,229,285 discloses the expression of a thermostable luciferase of a firefly.
Alteration of the bioluminescence emission of currently used luciferases can be obtained by amino acid substitutions and other mutations in the active sites of the luciferase genes. Although a number of such luciferases are available commercially, additional luciferase derivatives with altered spectral properties would be desirable.